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小鼠GITRL基因腺病毒载体的构建与鉴定 被引量：1

Construction and identification of recombinant adenovirus vector pAdEasy-GFP-GITRL
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摘要 目的:构建重组腺病毒pAdEasy-GFP-GITRL,并测定病毒滴度。方法:从PIRES-GITRL载体上用BglⅡ和SalⅠ双酶切,回收目的基因GITRL,插入同样用BglⅡ和SalⅠ双酶切的穿梭质粒pAdtrack-CMV中,将线性化穿梭质粒骨架质粒pAdEasy-1共转染BJ5183菌,鉴定正确后,将重组腺病毒载体pAdEasy-GFP-GITRL转染HEK293细胞,以包装出完整腺病毒。TCID50法测定重组腺病毒滴度,Western blot法检测GITRL蛋白表达。结果:成功构建小鼠GITRL基因的重组腺病毒载体(pAdEasy-GFP-GITRL),经包装后获得高滴度表达GITRL基因的重组腺病毒,病毒滴度为2.0×109pfu/mL。Western blot结果显示重组病毒载体能表达GITRL蛋白。结论:重组腺病毒表达载体(pAdEasy-GFP-GITRL)的成功构建及重组腺病毒的获得为GITRL基因干预治疗奠定了基础。 AIM： To construct recombinant adenovirus vector pAdEasy-GFP-GITRL and detect the viral titer.METHODS： GITRL gene was obtained by double digestion using BglⅡand SalⅠ,and cloned into the baculovirus transfer vector（pAdtrack-CMV）,then the recombinant adenovirus vector（pAdtrack-CMV-GITRL） was digested by restrictive endoenzyme PmeⅠ.The linear recombinant adenorirus vector and pAdEasy-1 were cotransfected into HEK293 cells by co-precipitate of calcium phosphate.Recombinant adenovirus was packaged and purified in HEK293A cells.RESULTS： Recombinant adenovirus vector pAdEasy-GFP-GITRL was constructed successfully and high titer of recombinant adenovirus was obtained（2.0×109 pfu/mL）.Western blotting analysis also revealed the expression of GITRL by recombinant adenovirus vector.CONCLUSION： The construction of recombinant adenovirus vector pAdEasy-GFP-GITRL and recombinant adenovirus will facilitate the potential GITRL gene therapy.
作者 马洁 田洁 刘艳 刘青玲 毛朝明 焦志军 许化溪 王胜军
机构地区 江苏大学免疫学和免疫检验学系 江苏大学附属医院
出处 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2010年第11期1075-1077,共3页 Chinese Journal of Cellular and Molecular Immunology
基金 国家自然科学基金资助项目(30871193 30972748 30910103087 81072453) 江苏省卫生厅科研基金资助项目(H2009052) 江苏省教育厅自然科学基金资助项目(08KJB320002) 江苏大学"拔尖人才"工程项目(2008年)
关键词 GITRL 重组腺病毒 病毒滴度 GITRL recombinant adenovirus viral titer
分类号 R392.12 [医药卫生—免疫学]
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参考文献7

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细胞与分子免疫学杂志
2010年 第11期

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